SERD), Has Antitumor Activity in Multiple Erþ Breast Cancer Patient-Derived Xenograft Models Teeru Bihani, Hitisha K

Published OnlineFirst May 4, 2017; DOI: 10.1158/1078-0432.CCR-16-2561

Cancer Therapy: Preclinical Clinical Cancer Research Elacestrant (RAD1901), a Selective Estrogen Receptor Degrader (SERD), Has Antitumor Activity in Multiple ERþ Breast Cancer Patient-derived Xenograft Models Teeru Bihani, Hitisha K. Patel, Heike Arlt, Nianjun Tao, Hai Jiang, Jeffrey L. Brown, Dinesh M. Purandare, Gary Hattersley, and Fiona Garner

Abstract

þ Purpose: Estrogen receptor–positive (ER ) breast cancers are Results: Elacestrant induces the degradation of ER, inhibits þ typically treated with endocrine agents, and dependence on the ER ER-mediated signaling and growth of ER breast cancer cell pathway is often retained even after multiple rounds of anties- lines in vitro and in vivo,andsignificantly inhibits tumor growth trogen therapy. Selective estrogen receptor degraders (SERD) are of multiple PDX models. Furthermore, we demonstrate that being developed as a strategy to more effectively target ER and elacestrant in combination with palbociclib or everolimus can exploit ER dependence in these cancers, which includes inhibiting lead to greater efficacy in certain contexts. Finally, elacestrant both wild-type and mutant forms of ER. The purpose of this study exhibits significant antitumor activity both as a single agent and was to evaluate the efficacy of a novel orally bioavailable SERD, in combination with palbociclib in two patient-derived breast þ elacestrant (RAD1901), in preclinical models of ER breast cancer. cancer xenograft models harboring ESR1 mutations. Experimental Design: Elacestrant was evaluated as a single Conclusions: These data underscore the potential clinical agent and in combination with palbociclib or everolimus in utility of elacestrant as a single agent and as a combination þ þ multiple ER breast cancer models, including several patient- therapy, for both early- and late-stage ER disease. Clin Cancer Res; derived xenograft models. 1–12. 2017 AACR.

Introduction retained (5–7). On the basis of this continued dependence on ER, novel selective estrogen receptor degraders (SERD) have gained Breast cancer is subdivided into categories based on the tumor widespread attention as a means of delivering more durable receptor status; speciﬁcally, whether the tumor expresses estrogen þ responses and increasing progression-free survival in this setting receptor (ER), progesterone receptor (PR) or Her2, with ER (8–11). disease making up the majority of patients in the breast cancer Currently, fulvestrant (Faslodex, AstraZeneca) is the only patient population (1). Given the dependence on ER in this approved SERD for the treatment of postmenopausal women disease segment, most treatment modalities focus on inhibiting þ with advanced ER breast cancer who have progressed on endo- various aspects of this pathway. Indeed, inhibition of estrogen crine therapies (http://www.accessdata.fda.gov/drugsatfda_docs/ synthesis (e.g., with aromatase inhibitors (AI)) or modulation label/2011/021344s015lbl.pdf). In the metastatic setting, fulves- of ER pathway activity (e.g., with tamoxifen), continue to be þ trant has been shown to be effective as a single agent and a recent mainstays in the standard of care for ER breast cancer patients study demonstrated that the increased dose of 500 mg fulvestrant both in the adjuvant and metastatic setting (ref. 2; https://www. is superior to the historical 250 mg dose in terms of overall nccn.org/professionals/physician_gls/pdf/breast.pdf). While survival (8, 12, 13). However, there is evidence to suggest that many patients with advanced and metastatic breast cancer initially even at the 500 mg dose, suboptimal occupancy of the estrogen respond to these agents, a majority of patients will have progres- receptor can occur in some patients, which may correlate with an sive disease (3, 4). Interestingly, even in these patients who have earlier progression of disease (14). These data, combined with seen multiple prior endocrine therapies, dependence on ER for fulvestrant's intramuscular route of administration underscore tumor growth and sensitivity to other ER-targeting agents is often the need for a novel orally bioavailable SERD for the treatment of þ ER breast cancer. Several reports have shown that the PI3K/mTOR and Rb/E2F Radius Health, Inc, Waltham, Massachusetts. signaling pathways can compensate for and crosstalk with the ER Note: Supplementary data for this article are available at Clinical Cancer signaling pathway, contributing to endocrine treatment resistance Research Online (http://clincancerres.aacrjournals.org/). (15–18). Given the activation of these growth- and survival- Corresponding Author: Fiona Garner, Radius Health, 950 Winter Street, promoting pathways after endocrine therapy, current clinical Waltham, MA 02451. Phone: 617-551-4000; Fax: 617-551-4701; E-mail: treatment strategies have begun combining endocrine-based [email protected] agents with small-molecule inhibitors of these pathways doi: 10.1158/1078-0432.CCR-16-2561 (19, 20). The increase in progression-free survival demonstrated 2017 American Association for Cancer Research. in clinical studies such as BOLERO-2 and PALOMA-1 where an

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elacestrant has potent antitumor effects in two PDX models that Translational Relevance harbor ESR1 mutations. Taken together, these results provide þ Advanced ER breast cancers often retain a dependence on strong preclinical data and mechanistic rationale for further ER and maintain sensitivity to multiple rounds of endocrine clinical investigation of elacestrant. therapy. Nevertheless, treatment resistance and progressive disease remain a clinical challenge and have been attributed Methods to ESR1 mutations and/or the activation of compensatory pathways. Here, we demonstrate the preclinical efficacy of Reagents and cell lines elacestrant, a novel SERD that is under clinical investigation Elacestrant (6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N- þ for the treatment of advanced ER breast cancer. Using mul- ethylamino)-4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen- tiple patient-derived xenograft (PDX) models, including some 2-ol dihydrochloride) was manufactured by Patheon. Elacestrant derived from heavily pretreated patients, we demonstrate the lots used in this study were periodically checked to ensure purity, efficacy of elacestrant as a single agent and in combination stability and chirality. with palbociclib or everolimus. Importantly, elacestrant was MCF7, T47D, and HCC1428 cells were purchased from ATCC also effective at inhibiting the growth of PDX models harbor- and were routinely maintained in phenol red-free minimal essen- ing ESR1 mutations. This study provides rationale for the tial medium (MEM) or RPMI medium supplemented with 0.01 þ ongoing clinical investigation of elacestrant in ER breast mg/mL bovine insulin and 10% FBS (HyClone, GE Healthcare cancer patients, both as a single agent and in combination Life Sciences) at 5% CO2. Cells were authenticated using STR with approved agents, such as palbociclib and everolimus. analyses and confirmed to be mycoplasma-free using the MycoA- lert kit (Lonza). Charcoal-dextran–stripped FBS was obtained from Gemini Bio and HyClone (GE Healthcare Life Sciences).

aromatase inhibitor was combined with everolimus (mTOR In vitro cancer cell proliferation inhibitor) or palbociclib (CDK4/6 inhibitor), respectively, high- The 50% growth inhibition concentration (IC50) of elacestrant lights the effectiveness of this strategy (21–23). Furthermore, was determined in MCF7 and T47D using the CellTiter-Glo recent data from the PALOMA-3 trial validate the use of a SERD luminescent cell viability assay (Promega). Briefly, cells were (fulvestrant) in combination with CDK4/6 inhibitors as a poten- incubated in phenol-red free media with charcoal-stripped FBS tial new treatment strategy for metastatic disease (24). for 72 hours before seeding into 96-well plates. Twenty-four hours While combinations have demonstrated superior efficacy com- postplating, cells were treated with increasing doses of elacestrant pared with the use of aromatase inhibitors alone, recent studies in the presence of 0, 0.01 nmol/L, and 1 nmol/L 17b-estradiol. have also demonstrated that mutations in the ESR1 gene itself can Cells were incubated in the indicated treatment for 7 days and the also drive estrogen-independent activity leading to clinical treat- CellTiter-Glo assay (Promega) was performed as per manufac- ment resistance (25–31). It has been reported that patients treated turer's instructions. with AIs are more likely to develop tumors harboring ESR1 mutations, resulting in ligand-independent transcriptional activ- Western blots ity of ER (27, 32). In addition, some ESR1 mutations can lead to For analysis of ER protein expression in response to treatment, conformational changes of ER, leading to decreased binding of MCF7, T47D, or HCC1428 cells were seeded in 6-well plates tamoxifen, thereby potentially reducing its activity (29, 31). These containing phenol-red free media supplemented with 10% char- findings highlight the need for a novel SERD that can bind and coal-stripped FBS. Forty-eight hours later, cells were treated with inhibit both wild-type and mutant forms of ER in order to more elacestrant or fulvestrant at the indicated concentrations. Cells effectively treat this patient population. Recent clinical data have were harvested and lysed in CellLytic M lysis buffer (Sigma- demonstrated that while the presence of ESR1 mutations can be a Aldrich) after 24 or 48 hours of treatment, and total protein was prognostic indicator of AI resistance, the extent of fulvestrant separated by SDS-PAGE and transferred to membranes and efficacy is not influenced by ESR1 status (32, 33), suggesting immunoblotted using antibodies specific to the indicated pro- SERDs have therapeutic value in this clinical context. teins. For in vivo pharmacodynamic studies, end of study flash- The changing breast cancer treatment landscape together with frozen tumors were fractured using a cryoPREP instrument (Cov- þ emerging molecular mechanisms underlying ER disease provide aris) and pulverized tissue was lysed in Cell Lysis Buffer (Cell strong rationale for the further clinical development of an orally Signaling Technology). Total protein was analyzed by Western þ bioavailable SERD in patients with ER breast cancer. Elacestrant blot analysis as described above. Antibodies specifictoERa (Cell (RAD1901), an orally bioavailable, nonsteroidal small-molecule Signaling Technology, #13258; Santa Cruz Biotechnology, SERD, is currently under clinical investigation for the treatment of sc-543), PR (Cell Signaling Technology, sc-3153), and anti-vin- þ ER breast cancers in postmenopausal women (NCT02650817, culin (Sigma-Aldrich, V9131) as an internal control were used. NCT02338349). In a previous report, we demonstrated that elacestrant selectively binds and induces the degradation of ER Quantitative reverse transcriptase PCR analyses (34). Furthermore, we demonstrated that elacestrant antagonized For cell lines, quantitative reverse transcriptase PCR analysis estrogen-mediated uterotrophic effects and had bone protective (qRT-PCR) was performed using the Cells to CT kit (Life Tech- effects in a rat osteopenia model, suggesting a favorable profile for nologies) using TaqMan probes. For in vivo pharmacodynamic use in patients. Here, we evaluate elacestrant activity in multiple studies, end of study flash-frozen tumors were fractured using a þ þ ER breast cancer cell lines and ER patient-derived tumor xeno- cryoPREP instrument (Covaris). From pulverized tissue, total graft (PDX) models as a single agent, and in combination with RNA was extracted using the RNeasy Mini Kit (Qiagen) and palbociclib or everolimus. In addition, we also demonstrate qRT-PCR was performed using the 1-step RNA to CT (Carlsbad)

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using TaqMan probes. The CT values were analyzed to assess tubes and processed for pharmacokinetic analysis. Analysis of relative changes in expression of the TFF1 (trefoil factor 1/breast fulvestrant in mouse plasma samples was carried out using high- cancer estrogen-inducible protein), GREB1 (gene regulated by performance liquid chromatography on a Pursuit XRs 3 Diphenyl estrogen in breast cancer 1) and PGR (progesterone receptor) 100 2.0 mm column (Agilent). DDC genes, with GAPDH as an internal control, using the 2 T method (35). IHC IHC was performed using the Leica Bond III system (Leica In vivo xenograft experiments Biosystems) according to the manufacturer's instructions. Primary All study protocols were reviewed by Radius, approved by rabbit mAbs were obtained from Cell Marque and were directed Institutional Animal Care and Use Committees (IACUC), and against HER2/Neu (SP3), Ki67 (D2H10), estrogen receptor (SP1), conducted in accordance with US and International regulations and progesterone receptor (SP42). for protection of laboratory animals. Female athymic nude mice (NU(NCr)-Foxn1nu or BALB/cAnNCrl-Foxn1nu) were obtained In vitro binding assays from Envigo RMS, Inc., Harlan Laboratories, or Charles River In vitro binding affinity of elacestrant was determined using Laboratories and acclimated for 3 to 7 days prior to implantation. purified ligand-binding domain of wild-type and mutant ERa in Mice were given water (reverse osmosis, 1 ppm Cl) and fed a daily the PolarScreen ERa Competitor Assay (Thermo Scientific, complete diet ad libitum, and were housed on irradiated bedding #A15883) as per manufacturer's instructions. on a 12- to 14-hour light cycle under controlled temperature and humidity. Preformulated, clinical-grade fulvestrant (Faslodex, Statistical and data analysis manufactured by AstraZeneca) was obtained through third party Statistical analysis and graphical presentations were performed vendors and administered by subcutaneous injection once week- using GraphPad Prism 6 or 7 and Microsoft Excel. Descriptive data ly. Elacestrant, palbociclib and everolimus were administered were generally expressed as mean SD or SEM. Statistical evalua- daily by oral gavage. In the ST941 study, groups receiving palbo- tions of the differences between groups were assessed using ciclib were initially administered 100 mg/kg and dose reduced to Student t test analyses, with a prespecified alpha of 0.05. For cell 75 mg/kg on day 14 of treatment. At the end of this study, average proliferation assays, the IC50 was calculated by fitting a body weight loss for all treatment groups did not exceed 15%. dose–response curve using a nonlinear regression model with a sigmoidal dose response. The formula for calculating surviving MCF-7 xenografts. Twenty-four hours prior to implantation of rate is shown below; Relative IC50 is calculated where y-axis was set MCF-7 cells, estrogen pellets (0. 18 mg/pellet 17bestradiol, at 50% using GraphPad Prism 5.0 curve fitting software. For all 90-day release, Innovative Research of America) were implanted tumor xenograft models, body weights and tumor volumes subcutaneously between the scapulae of female athymic nude were evaluated twice weekly. Tumor volume was calculated 6 mice using a sterilized trochar. MCF7 cells (5 10 per mouse) in using the formula: tumor volume (mm3) ¼ 0. 5 L W2, where 50:50 Matrigel (Corning Inc.):MEM were implanted in the rear W ¼ width and L ¼ length in mm of the tumor. Relative tumor fl ank. When mean tumor volumes reached approximately 150 to volumes were defined as tumor volumetreatment day X tumor 3 200 mm , mice were randomized to treatment groups based on volumetreatment day 0. Tumor growth inhibition (%TGI) was tumor size. For pharmacodynamic analyses, MCF7 xenograft- calculated as (1(average relative tumor volumetreatment group/ bearing mice were treated daily for seven days, animals were average relative tumor volumevehicle group))100). Tumor volume euthanized, and tumors collected 4 and 24 hours post-last dose. data were analyzed using a linear mixed-effects model with log10 transformation of postbaseline value over baseline as response Patient-derived xenograft models. HBCx-21, HBCx-3 and HBCx-19 variable, treatment, day, and treatment-by-day interaction as fixed patient-derived tumor xenografts (PDX) were derived at and effects, and animal as random effect assuming a first-order auto- studies run at XenTech. The ST986, ST941, and ST2177 PDX regression correlation between days for a specific animal. Treat- models were derived at and studies run at South Texas Accelerated ment group comparisons were performed using least square Research Therapeutics (San Antonio, TX). MAXF-713 was derived means of the log10 transformed postbaseline value over baseline at and studies run at Charles River Discovery (OncoTest). All at the end of study, with P values adjusted using the Bonferroni animals were subcutaneously implanted with PDX models and method, a conservative P value adjustment method (ns, not began receiving estrogen supplementation in the drinking water significant; , P < 0.05; , P < 0.01; , P < 0.001; Supplementary from the date of tumor implant to the end of the study. The HBCx- Table). 19, HBCx-3, and HBCx-21 (XenTech) models were supplemented b with 8.5 milligrams of 17 -estradiol to each liter of drinking Results water. The MAXF-713 model (Charles River Discovery, Oncotest) was supplemented with 10 milligrams of 17b-estradiol to each Elacestrant is a SERD that inhibits breast cancer cell liter of drinking water. When tumors grew to 150–200 mm3, mice proliferation in vitro and in vivo were randomized on the basis of tumor volume and administered To determine the ER degrading activity of elacestrant in the the indicated treatments. At the end of study, tumors were breast cancer setting, ER protein expression was examined in þ harvested 4 hours post-last dose unless otherwise indicated. multiple ER breast cancer cell lines (MCF7, T47D, and HCC1428) following treatment with increasing doses of elaces- In vivo pharmacokinetic analyses trant or fulvestrant. Elacestrant treatment resulted in a dose- Terminal plasma was collected via heart puncture and nonter- dependent and marked decrease in ER protein expression, induc- minal plasma was collected via orbital bleeding. For all mice, ing degradation of ER to a similar extent as fulvestrant in all three blood samples were collected in potassium-EDTA–containing cell lines tested (Fig. 1A). In addition, elacestrant treatment

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Elacestrant Inhibits Growth of ERþ Breast Cancer PDX Models

resulted in decreased expression of progesterone receptor (PGR, combination with either palbociclib or everolimus (Fig. 2C PR), an ER target gene, in both MCF7 and T47D cell lines (Fig. 1B; and D). Of note, a lower dose of elacestrant (30 mg/kg) elicited Supplementary Fig. S1A). Regardless of the 17b-estradiol (E2) similar effects on tumor volume as a single agent and in combi- concentration used (10 pmol/L or 1 nmol/L representative of a nation with palbociclib or everolimus (Supplementary Fig. S4). postmenopausal and premenopausal setting, respectively; Similarly, the %TGI observed with fulvestrant in combination ref. 36), elacestrant was able to decrease PGR to a basal level with palbociclib or everolimus was greater than fulvestrant alone; similar to control cells grown in the absence of E2 (Fig. 1B; however, the tumor volume change was not significantly different Supplementary Fig. S1A). Importantly, elacestrant did not between these treatment groups (Supplementary Table). increase ER target genes in the absence of estradiol, suggesting ER and PR protein expression was assessed in individual tumors elacestrant has an antagonistic profile in the breast cancer setting at the end of study and both proteins were decreased in all (Supplementary Fig. S1). Analysis of additional ER target genes, elacestrant-treated groups, including the combination groups TFF1 and GREB1, revealed similar effects following elacestrant (Fig. 2E and F). Interestingly, everolimus treatment appeared to treatment (Supplementary Fig. S1B). Consistent with the ability of increase ER expression, and despite this increase, elacestrant was elacestrant to induce ER degradation and block ER signaling, able to induce ER degradation, suggesting elacestrant effectively elacestrant treatment also inhibited E2-mediated proliferation in counteracts feedback pathways that can induce ER expression MCF7 and T47D cells in a dose-dependent manner (Fig. 1C). (Fig. 2F). These findings confirm the ability of elacestrant to effectively induce ER degradation and inhibit estrogen-mediated signaling Elacestrant demonstrates significant antitumor efficacy in þ and growth of multiple ER cell lines. multiple PDX models þ We further evaluated the in vivo activity of elacestrant as a single The molecular classification of ER disease in the clinical agent in the MCF7 cell line xenograft model. Following once daily setting is routinely determined using IHC staining, where a tumor oral administration of either 30 mg/kg or 60 mg/kg, elacestrant can be designated ER-positive with as few as 1% of cells within the þ induced complete tumor growth inhibition after four weeks of tumor expressing ER (37). Patients who have ER tumors, regard- treatment (Fig. 1D). Of note, the plasma concentration of these less of the extent of ER positivity, are typically treated with elacestrant doses are clinically achievable (manuscript in prepa- endocrine agents (37, 38). To more closely mimic the tumor ration). Fulvestrant treatment also resulted in tumor growth heterogeneity of ER in the clinical setting, we evaluated effects of þ inhibition in this model following a 3 mg/week dose (Fig. 1D). elacestrant and fulvestrant on five ER PDX models with varied We determined the 3 mg/week dose of fulvestrant in mice ER/PR expression levels (Table 1). achieved a plasma concentration approximately 1.8-fold higher Three of the models chosen, ST986 (ESR1:WT, PIK3CA:E542K), than that of the approved 500 mg clinical dose (Supplementary HBCx-21 (ESR1:WT, PIK3CA:WT), and MAXF-713 (ESR1:WT, Fig. S2). Importantly, at the end of the study, body weight loss did PIK3CA:WT), were selected on the basis of relatively high expres- not exceed 10% in any of the treatment groups suggesting both sion levels of wild-type ER and PR (Table 1; Supplementary Fig. elacestrant and fulvestrant were well-tolerated (Supplementary S5A). Importantly, each model displayed differential sensitivity to Fig. S3A). To further understand treatment effects, tumor volumes fulvestrant (1 mg/week), a dose that we determined achieved an were evaluated after treatment had been withdrawn. Interestingly, equivalent plasma exposure as the 500 mg clinical dose (Supple- tumor growth inhibition was maintained for four weeks after mentary Fig. S2). This dose of fulvestrant was sufficient to induce elacestrant or fulvestrant withdrawal (Fig. 1D, inset), suggesting complete tumor growth inhibition of the HBCx-21 model, where- these treatments resulted in sustained tumor growth inhibition. as it resulted in partial growth inhibition of the MAXF-713 model To better understand the pharmacodynamic effects on the ER and did not result in significant tumor growth inhibition in the pathway, downstream ER signaling was examined in MCF7 ST986 model (Fig. 3A–C). Interestingly, increasing the fulvestrant tumors treated with elacestrant for seven days. Notably, PR dose to 5 mg/week, equivalent to approximately 2.9 times the expression was significantly decreased in all elacestrant-treated clinical exposure (Supplementary Fig. S2), resulted in growth groups and this decrease was maintained up to 24 hours following inhibition of ST986 tumors (Fig. 3A). Elacestrant at all doses the seventh dose (Fig. 1E; Supplementary Fig. S3). These results tested (30 mg/kg, 60 mg/kg and 120 mg/kg), consistently resulted demonstrate that elacestrant can produce a stable and continued in significant tumor growth inhibition in all three PDX models pharmacodynamic effect on ER signaling and sustained tumor (Fig. 3A–C). growth inhibition across a range of doses. Tumors collected at the end of treatment were assessed for changes in ER and PR expression. Consistent with the results seen Elacestrant treatment results in tumor regressions in MCF7 in MCF7 tumors (Figs. 1 and 2), elacestrant treatment decreased tumor xenografts as a single agent and in combination with ER protein expression in all three models and this was accom- palbociclib or everolimus panied by a decrease in PR (Fig. 3D; Supplementary Fig. S5B and To support our hypothesis that elacestrant would be an effective S5C). endocrine backbone for combination strategies, MCF7 tumor HBCx-21–treated tumors were also examined following treat- xenografts were treated with elacestrant (60 mg/kg) as a single ment withdrawal in animals treated with the lowest 30 mg/kg agent or in combination with either everolimus or palbociclib. dose of elacestrant, or 1 mg/week of fulvestrant. Similar to the The combination of elacestrant with either everolimus or palbo- result in MCF7 xenografts, tumor growth inhibition in the ela- ciclib resulted in significantly greater tumor growth inhibition as cestrant treatment group and the fulvestrant treatment group was compared with elacestrant single-agent effects (Fig. 2A and B). maintained for almost two months posttreatment (Supplemen- Evaluation of the changes in tumor volume pre- and posttreat- tary Fig. S5D). Furthermore, analysis of the limited tumor mate- ment revealed that elacestrant treatment produced tumor regres- rial that remained displayed sustained decreases in ER expression sions in the majority of animals when used as a single agent or in in the elacestrant and fulvestrant-treated tumors, despite lack of

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A 1,200 B 1,200 Vehicle Vehicle Palbociclib 45 mg/kg Everolimus 2.5 mg/kg 1,000 Fulvestrant 3 mg/dose 1,000 Fulvestrant 3 mg/dose Fulvestrant + palbociclib Fulvestrant + everolimus Elacestrant 60 mg/kg Elacestrant 60 mg/kg ) ) 3 3 Elacestrant + everolimus 800 Elacestrant + palbociclib 800

600 600

400 ns 400 Tumor volume (mm ** Tumor volume (mm *** *** *** 200 200 * ***

0 0 0203010 0203010 Days of treatment Days of treatment C D 1,200 Individual tumors, end of study 1,200 Individual tumors, end of study

500 500

Fulvestrant Fulvestrant + + 300 palbociclib 300 everolimus

Elacestrant Elacestrant + + 100 palbociclib 100 everolimus % Change from baseline % Change from baseline

-100 -100 Vehicle Fulvestrant Palbociclib Fulvestrant/ Elacestrant Elacestrant/ Vehicle Fulvestrant Everolimus Fulvestrant/ Elacestrant Elacestrant/ palbociclib palbociclib everolimus everolimus

Elacestrant Elacestrant + + E Vehicle Elacestrant Palbociclib palbociclib F Vehicle Elacestrant Everolimus everolimus ER ER

PR PR

Vinculin Vinculin

Fulvestrant Fulvestrant + + Vehicle Fulvestrant Palbociclib palbociclib Vehicle Fulvestrant Everolimus everolimus

ER ER

PR PR

Vinculin Vinculin

Figure 2. Elacestrant drives tumor regressions alone and in combination with approved clinical agents in MCF7 xenografts. Animals bearing MCF7 tumors were treated for 27 days with elacestrant alone, fulvestrant alone, or either SERD in combination with palbociclib or everolimus. A and B, Mean tumor volumes (n ¼ 9–10/arm) SEM. Asterisks represent signiﬁcant differences between the indicated treatment groups on day 27. C and D, Percent change in tumor volumes from individual animals from start of treatment to end of treatment. E and F, Representative Western blots from individual tumors from A–D collected 2 hours posttreatment on day 27. Data representing single agent vehicle-, elacestrant-, and fulvestrant-treated animals are duplicated in A and B, C and D, and E and F.

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Elacestrant Inhibits Growth of ERþ Breast Cancer PDX Models

Table 1. Characterization of PDX models ER ER PR PR Model Patent Rx-history (Intensity) (% positive cells) (Intensity) (% positive cells) Her2 ST986 Rx-na€ve 2 84 2 77 þ MAXF-713 Rx-na€ve 3 98 3 85 HBCx-21 Rx-na€ve 3 99 3 98 HBCx-3 Rx-na€ve 2 61 3 51 þ HBCx-19 Rx-na€ve 2 34 3 8 þ ST2177 (ESR1:Y537Shom) tam, AI 2 45 3 83 þ ST941 (ESR1:Y537Shet) AI 2 52 3 73 þ NOTE: Patient Rx-history refers to therapies administered to patients prior to development of PDX. IHC analyses on vehicle-treated tumors using antibodies speciﬁc for human ER, PR and Her2. Mean of %positive cells shown (n ¼ 3–6 tumors/model analyzed). Abbreviations: AI, aromatase inhibitor; Rx, treatment; Tam, tamoxifen.

SERD treatment for an extended period (Supplementary Fig. S5E). We also evaluated the effect of elacestrant and fulvestrant in These results conﬁrm the potent and stable antitumor efﬁcacy of PDX models expressing intermediate (HBCx-3) and low (HBCx- elacestrant in clinically relevant patient-derived tumor models 19) expression levels of ER and PR (Table 1; Supplementary Fig. expressing high levels of ER signaling and further validate that the S5A). On the basis of IHC analyses, approximately 60% of cells elacestrant-mediated biological effects extend beyond cell line– expressed ER and 50% of cells expressed PR in the HBCx-3 model derived tumor xenograft models. (Table 1). In HBCx-3, both fulvestrant and elacestrant resulted in

A ST986 B MAXF-713 C HBCx-21 84% ER+, 77% PR+ 98% ER+, 85% PR+ 99% ER+, 98% PR+ 1,600 1,000 Vehicle 1,500 Vehicle Vehicle Fulvestrant 1 mg/dose Fulvestrant 1 mg/dose Fulvestrant 1 mg/dose ) 1,400 3 Fulvestrant 5 mg/dose 1,250 Elacestrant 30 mg/kg Elacestrant 30 mg/kg ns ) ) Elacestrant 30 mg/kg 3 3 800 1,200 Elacestrant 60 mg/kg Elacestrant 60 mg/kg Elacestrant 60 mg/kg 1,000 Elacestrant 120 mg/kg 1,000 600 800 750 600 400 500 *** Tumor volume (mm *** 400 *** Tumor volume (mm *** Tumor volume (mm 200 250 *** 200 *** *** *** 0 0 0 05 101520253035404550556065 50 10 15 20 25 30 35 40 45 50 55 60 05 1015202530354045505560 Days of treatment Days of treatment Days of treatment

D E HBCx-3 + Fulvestrant Elacestrant 61% ER, 51% PR 1 mg Vehicle 5 mg 30 mg/kg 60 mg/kg Vehicle 1,600 Fulvestrant 1 mg/dose ER Elacestrant 30 mg/kg

) Elacestrant 60 mg/kg 3 1,200 Elacestrant 120 mg/kg ** ST986 *** PR 800 *** ***

400 Tumor volume (mm Elacestrant VehicleFulvestrant 30 mg/kg 60 mg/kg 0 05 1015202530354045505560 ER Days of treatment

HBCx-21 PR

Figure 3. Elacestrant displays potent anti-tumor activity in multiple PDX models. A–C, Mean tumor volumes SEM of indicated PDX models treated with elacestrant or fulvestrant at the indicated doses. ST986, n ¼ 6/arm. MAXF-713 and HBCx-21, n ¼ 9–10/arm. Asterisks represent significant differences of the indicated treatment groups relative to vehicle control at end of treatment. D, IHC analyses of tumors harvested 4 hours posttreatment at the end of study from ST986 (top) and HBCx-21 (bottom) using antibodies specific for ER or PR. Representative photomicrographs shown (40 magnification). E, Mean tumor volumes (n ¼ 10/arm) SEM of HBCx-3 model. Asterisks represent significant differences of the indicated treatment groups relative to vehicle control on day 42.

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significant, yet partial growth inhibition (Fig. 3E) with a corre- These data suggest elacestrant can induce greater %TGI in a model sponding decrease in PGR mRNA (Supplementary Fig. S5F). with higher ER and PR expression (Table 1; Supplementary In both HBCx-3 (61% ER, 51% PR) and MAXF-713 (98% ER, Fig. S5A) and that models with intermediate ER and PR expression 85% PR), the addition of fulvestrant to elacestrant did not achieve might benefit from combination strategies. a significant change in tumor volume compared with elacestrant as a single agent, suggesting elacestrant alone achieves maximal Elacestrant effectively inhibits growth of PDX models efficacy at the doses tested in these models (Supplementary Fig. harboring ESR1 mutations S6A and S6B). Furthermore, neither compound was efficacious in As mentioned above, patients who have received multiple the HBCx-19 model (Supplementary Fig. S6C). The IHC results rounds of endocrine therapies often progress, yet many main- demonstrated that 34% of cells expressed ER and 8% of cells tain sensitivity to ER-targeting agents (7). This is further sup- expressed PR in the HBCx-19 tumors (Table 1), suggesting the ER ported by data demonstrating elacestrant displays single-agent signaling pathway may play a less significant role in driving tumor efficacy in multiple models that were derived from heavily growth in this model. In addition to the SERDs tested, tamoxifen pretreated patients that received prior endocrine therapies was also ineffective at inhibiting tumor growth in the HBCx-19 (Supplementary Fig. S7). The emergence of clinical ESR1 muta- model, confirming the lack of efficacy seen in this model is tions has recently gained attention as an increasingly important consistent across different classes of ER-targeting agents (Supple- mechanism of clinical resistance to endocrine therapies (25– mentary Fig. S6C). Collectively, these data suggest that elacestrant 31). To test the potential for elacestrant to inhibit the growth of acts in vivo maximally to inhibit ER signaling and tumor growth in ESR1-mutant tumors, we utilized two PDX models (ST2177 these PDX models. Furthermore, these data also demonstrate that and ST941) that harbor a Y537S mutation in the ESR1 gene PDX models with lower levels of ER and PR expression were (Table 1). This specific mutation is among the most frequently generally less sensitive to antiestrogens. reported aberrations found in tumors from patients treated To explore the correlation between ER/PR and sensitivity to with aromatase inhibitors (27), and has also been shown to endocrine agents further, we performed an efficacy screen with result in high levels of constitutive ER signaling. Consistent elacestrant in eight PDX models derived from patients that were with this, these two ESR1 mutant PDX models were derived clinically diagnosed with luminal A or luminal B breast cancers. from patients who were treated with aromatase inhibitors and The models used for the screen were confirmed for ER, PR, and the models were confirmed to have high levels of PR expression Her2 expression and in large part retained similar ER and PR levels (Table 1; Supplementary Fig. S5A). as described in the clinical diagnoses (Supplementary Fig. S7). The in vivo efficacy of elacestrant and fulvestrant was examined These models represented a wide range of ER and PR levels, with to evaluate the relative efficacy of these agents in tumors expres- the assumption that models with higher ER/PR expression would sing ESR1 mutations. Fulvestrant at 3 mg/dose did not result in a rely more heavily on the ER pathway for growth, and therefore be significant decrease in tumor volumes in the ST941 model and the more susceptible to ER-antagonism (Supplementary Fig. S7). 1 mg/dose was partially effective in the ST2177 model (Fig. 5A– Indeed, the in vivo screen validated our hypothesis demonstrating D). It should be noted, however, higher doses of fulvestrant were that models with higher ER and PR expression generally displayed not tested in these models; therefore, we cannot determine increased sensitivity to elacestrant. While ER signaling alone is whether a greater efficacy could have been achieved. Elacestrant unlikely to be the sole determinant for single-agent activity, these induced complete growth inhibition of ST2177 tumors at both results further corroborated our findings from the initial PDX doses tested (30 mg/kg and 60 mg/kg), and demonstrated a dose- studies (Fig. 3; Supplementary Fig. S7). dependent growth inhibition in ST941 tumors (Fig. 5A–D). The addition of palbociclib to elacestrant (60 mg/kg) resulted in a Correlation of hormone receptor status and combination significant decrease in tumor volumes relative to elacestrant alone efficacy in ST941, suggesting an additional benefit may be realized in The aforementioned correlation with ER status led us to further some patients with ESR1-mutant tumors using a combination hypothesize that PDX models with intermediate ER and PR strategy. expression may represent a setting that would benefit from PR expression was also examined at the end of treatment in combinations with approved agents. To this end, we treated these two models using both IHC and qRT-PCR (Fig. 5E and F). HBCx-3 tumors with elacestrant in combination with palbociclib Interestingly, elacestrant resulted in a dose-dependent decrease of or everolimus. Interestingly, while the combination of elacestrant PR in both models. This result was consistent in the ST941 model with fulvestrant did not result in greater efficacy compared with as a corresponding dose-dependent efficacy was also observed. elacestrant alone (Supplementary Fig. S6A and S6B), the combi- Interestingly, in ST2177, a dose-dependent decrease in PR was nation of elacestrant with either palbociclib or everolimus in the observed despite complete %TGI observed with both doses of HBCx-3 model resulted in significantly greater efficacy than any elacestrant (Fig. 5E and F). single-agent alone (Fig. 4A and B). As a comparison, combina- Together, these results further demonstrate the potent in vitro tions of elacestrant with palbociclib or everolimus were also and in vivo activity of elacestrant to inhibit ER expression, down- þ examined in the MAXF-713 PDX model that expressed relatively stream signaling and ER-mediated growth of multiple ER breast high levels of ER and PR. In this model, elacestrant treatment cancer models. These data also suggest elacestrant has consistent alone resulted in a significant decrease in tumor volume relative to and maximum inhibitory effects on the ER pathway, and that the vehicle treatment (Fig. 3B; Supplementary Fig. S6). The addition combination of elacestrant with other targeted therapies can of everolimus to elacestrant did not significantly decrease tumor provide additional benefit. Finally, we have demonstrated that volumes and the addition of palbociclib to elacestrant resulted in elacestrant is capable of producing regressions in tumor models a modest, yet significant decrease in tumor volumes compared derived from heavily pretreated patients including those harbor- with elacestrant alone in this model (Supplementary Fig. S6D). ing ESR1 mutations.

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A HBCx-3 B HBCx-3 61% ER+, 51% PR+ 61% ER+, 51% PR+ Vehicle Vehicle 2,200 Elacestrant 60 mg/kg 2,200 Elacestrant 60 mg/kg Palbociclib 75 mg/kg Everolimus 2.5 mg/kg ) ) 3 Elacestrant + palbociclib 3 1,800 1,800 Elacestrant + everolimus

1,400 1,400

1,000 *** *** 1,000 *** *** 600 600 Tumor volume (mm Tumor volume (mm

200 200

0420 0600420 060 Days of treatment Days of treatment

Figure 4. Elacestrant in combination with palbociclib or everolimus in a PDX model. Mean tumor volumes (n ¼ 8–10/arm) SEM of HBCx-3 xenografts treated with elacestrant (60 mg/kg) and either palbociclib (A) or everolimus (B). Asterisks represent signiﬁcant differences between the indicated treatment groups on day 46.

Discussion (allowing target engagement of a SERM or SERD) and PR (suggesting dependence on the ER pathway signaling for growth and In this report, we describe an orally bioavailable SERD, elaces- survival). This is an important observation to consider, given that trant, and its potent growth-inhibitory activity in multiple models þ standard clinical practice recommends endocrine treatment for of ER breast cancer. We demonstrate that the in vitro effects of þ patients with ER tumors with as few as 1% of cells with the tumor elacestrant (ER degradation, ER signaling blockade, and growth expressing ER (37). Given the results presented here demonstrat- inhibition) translate to in vivo efficacy in multiple patient-derived ing minimal efficacy with ER-targeting agents in PDX models with tumor xenograft models (Table 1; Supplementary Fig. S8). Spe- low/no ER/PR expression, this concept warrants further investi- cifically, elacestrant as a monotherapy demonstrated efficacy in gation. Indeed, a recent retrospective study comparing patients attenuating the growth of multiple clinically relevant PDX mod- with tumors that were 1%–9% ER-positive versus greater than els, many of which were derived from patients who were heavily 10% ER positive suggested that the patients with lower ER pos- pretreated (Supplementary Figs. S7 and S8A). Moreover, in mod- itivity did not benefit from endocrine therapy (39). In fact, the lack els with high ER/PR expression, elacestrant demonstrated maxi- of response to endocrine therapies in these patients was similar to mal efficacy as a single agent, whereas in models expressing lower patients with basal (ER-negative) disease; however, it would be ER/PR levels a combination with palbociclib or everolimus was important to confirm this with a randomized clinical study. required to achieve similar efficacy (Supplementary Fig. S8B). Furthermore, the data in the HBCx-3 model suggest that a Furthermore, elacestrant treatment resulted in significant tumor model with intermediate levels of ER and PR is only partially growth inhibition in two PDX models that harbor ESR1 muta- sensitive to a SERD, and the addition of a second SERD did not tions (Table 1; Fig. 5). Together, these data suggest the potential þ result in additional efficacy at the doses tested (Supplementary clinical utility of elacestrant in the treatment of ER breast cancers, Fig. S6A). These data suggest that these tumors are likely driven by both in the early and late stages of disease. other pathways in addition to ER signaling. Our data demon- To date, fulvestrant remains the only SERD approved in the þ strating increased efficacy with combinations of elacestrant and clinic for the treatment of ER breast cancer. In the in vitro setting, palbociclib or everolimus in HBCx-3 confirm this hypothesis we demonstrate the ability of elacestrant to induce ER degradation (Fig. 4). Similarly, in the MAXF-713 model, the combination of to the same extent as fulvestrant and this corresponded to a fulvestrant and elacestrant did not significantly increase the %TGI blockade of ER signaling (Fig. 1). We further demonstrate elaces- compared with the %TGI with elacestrant alone. However, the trant is able to inhibit tumor growth similar to fulvestrant in in vivo combination of palbociclib and elacestrant resulted in a slight, yet MCF7 tumor xenografts. In this study, elacestrant demonstrated significant increase in %TGI compared with elacestrant alone, potent antitumor activity in multiple models at the clinically suggesting additional pathways can remain active despite high achievable and well-tolerated dose of 30 mg/kg. Importantly, at ER/PR levels. Collectively, these data demonstrate that (i) elaces- this dose, elacestrant achieved mouse plasma exposure similar trant can elicit potent antagonism on the ER pathway and (ii) to the human plasma exposure corresponding to a clinical dose elacestrant may serve as an effective endocrine backbone for that produces a robust estrogen receptor engagement based þ combinations with multiple agents in the treatment of ER breast on 16a-[18F]Fluoro-17b-estradiol (FES) uptake (manuscript in cancer. This idea is supported by the recent data from the preparation). PALOMA-3 trial, which demonstrated increased progression-free The data presented here suggest a correlation between survival when palbociclib was combined with fulvestrant com- the efficacy of an ER-targeting agent with the expression of ER pared with patients treated with fulvestrant alone (24). In

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A ST2177 B ST941 Vehicle Vehicle Fulvestrant 3 mg/dose 2,000 3,000 Palbociclib 75 mg/kg Fulvestrant 1 mg/dose Elacestrant 60 mg/kg Elacestrant 30 mg/kg Elacestrant 120 mg/kg ) ) 2,500 3 3 1,600 Elacestrant 60 mg/kg Elacestrant 60 mg/kg + palbociclib ns 2,000 1,200 *** *** 1,500 *** 800 *** 1,000 *** *** *** Tumor volume (mm Tumor volume (mm 400 500

0 0 010 2030405060 010 20304050 Days of treatment Days of treatment C D 1,200 ST2177 2,000 ST941 900 1,200 600 1,000 500 400 300 500 200 % Change from baseline

100 % Change from baseline 0 0 -100 Vehicle Fulvestrant Elacestrant Elacestrant Vehicle FulvestrantPalbociclib Elacestrant Elacestrant Elacestrant 30 mg/kg 60 mg/kg 60 mg/kg 120 mg/kg 60 mg/kg + palbociclib

E ST2177 ST941 F Elacestrant 1.5 1.5 ** **** **** Vehicle Fulvestrant 30 mg/kg 60 mg/kg ***

1.0 1.0 ST2177

0.5 0.5 Fold change Fold change Elacestrant VehicleFulvestrant 60 mg/kg 120 mg/kg

(relative to vehicle control) 0.0 (relative to vehicle control) 0.0

palbo ST941 Vehicle ctrl Vehicle mg/dose)ctrl

ElacestrantElacestrant (30 mg/kg) (60 mg/kg) Elacestrant Palbociclib(60 mg/kg) (75 mg/kg) Fulvestrant (1 mg/dose) FulvestrantElacestrant (3 (120 mg/kg)

Elacestrant (60 mg/kg) +

Figure 5. Elacestrant inhibits growth of two PDX models with Y537S mutations in ESR1. A and B, (top) Mean tumor volumes (n ¼ 8–10/arm) SEM of ST2177 (A) or ST941 treated with the indicated compounds at the indicated doses (B). For ST2177, asterisks represent significant differences between the indicated groups at the end of study. For ST941, statistical analyses of elacestrant-treated groups versus vehicle-treated groups were performed on the day that vehicle-treated tumors were harvested (day 21). Statistical analyses of fulvestrant-treated groups were compared with vehicle-treated groups on the day that fulvestrant-treated animals were harvested (day 17). C and D, Percent change in tumor volumes from individual animals at end of treatment for ST2177 and on the days in which ST941 were analyzed for statistical significance as outlined above. E, qRT-PCR analyses of PGR in ST2177 or ST941 tumors treated with elacestrant or fulvestrant at the indicated doses. One-way ANOVA was used to detect statistical significance between the indicated groups. , P < 0.01; , P < 0.001; , P < 0.0001. Histogram depicts the mean fold change (n ¼ 4) relative to the mean of vehicle control (n ¼ 4) SD. F, IHC of tumors collected 4 hours posttreatment from A and B using an antibody specific for PR. Representative photomicrographs shown (40 magnification).

summary, these results suggest ER/PR levels should be considered Consistent with clinical reports (40), the results presented here þ along with the heterogeneity of ER breast cancers, to provide suggest expression of progesterone receptor in addition to ER, patients with the most appropriate therapeutic strategies and to might have predictive value in determining antiestrogen treat- improve the chance of clinical success. ment response. While baseline PR levels in each model might be

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predictive of response, a complete decrease in PR after SERD potently inhibit the growth of PDX models derived from patients treatment might not be required for efficacy. For example, both previously treated with multiple endocrine therapies, including doses of elacestrant tested (30 mg/kg and 60 mg/kg) produced a two that harbor an ESR1 Y537S mutation (Fig. 5). Preliminary consistent and significant tumor growth inhibition in the ST2177 data suggests that elacestrant is able to bind to wild-type ESR1 and model; however, the lowest dose of elacestrant, 30 mg/kg, multiple mutant forms of ESR1 with similar potency in an in vitro appeared to have a limited effect on PR expression (Fig. 5E and binding assay, suggesting elacestrant could have activity against a F). It is possible that changes in ER target genes beyond PGR could broader panel of mutations beyond Y537S (Supplementary Fig. be more correlative with SERD response. Indeed, treatment of S10). Given that multiple clinical ESR1 mutations have been ST2177 tumors with 30 mg/kg elacestrant did result in a signif- identified, it will be interesting to continue to evaluate elacestrant icant decrease in TFF1 expression (Supplementary Fig. S9A). In a efficacy against these other ESR1 mutations. Finally, it will be second example, a decrease in PR expression was observed in important to track ESR1 allele burden in future clinical studies elacestrant treated MCF7 tumors 4 hours and 24 hours following when evaluating response to elacestrant, as many patients that are dosing (Supplementary Fig. S3C). When elacestrant-treated heavily pretreated with aromatase inhibitors have tumors with tumors were evaluated four weeks posttreatment, PGR expression multiple mutations in ESR1 (32). was no longer decreased, whereas GREB1 expression was The data presented here further describe the novel, orally decreased (Supplementary Fig. S9B). It will be important to bioavailable SERD, elacestrant, which potently inhibits ER-medi- þ understand specific pharmacodynamic changes that correlate ated signaling and growth of multiple ER breast cancer models. with sustained tumor growth inhibition in the context of long- Elacestrant significantly inhibits tumor growth of clinically rele- term treatment and posttreatment settings. Recently, a study vant patient-derived wild-type and mutant ESR1 xenograft mod- evaluating PR levels and response to fulvestrant demonstrated els as a single agent, and may serve as an effective endocrine that while initial PR levels indicated sensitivity to fulvestrant in backbone for use in combination therapy with other approved patients, the reduction in PR levels did not appear to correlate with agents. long-term efficacy (41). These data and reports suggest it will be Collectively, these results provide strong rationale for further important to evaluate a broader panel of ER target genes in clinical investigation of elacestrant in multiple settings, including response to elacestrant, with the notion that global ER antago- the potential treatment of endocrine na€ve patients, as well as þ nism might more accurately correlate with response to ER-target- heavily pretreated patients with ER breast cancer. ing agents. Interestingly, a recent report demonstrated that fulvestrant can effectively antagonize ER signaling and E2-dependent Disclosure of Potential Conflicts of Interest proliferation in the absence of ER degradation (42). This further All authors hold ownership interest in Radius Health, Inc. highlights the importance of global ER antagonism and its link to efficacy. It will be important to further understand the mechan- Authors' Contributions isms of ER degradation by elacestrant as it relates to the down- Conception and design: T. Bihani, J.L. Brown, D.M. Purandare, G. Hattersley, stream effects on tumor growth inhibition. Finally, it will be F. Garner interesting to determine whether the ER target gene profile and Development of methodology: T. Bihani, H. Arlt, N. Tao, H.K. Patel Acquisition of data (provided animals, acquired and managed patients, the ER coactivator/corepressor recruitment might be different in provided facilities, etc.): T. Bihani, H. Arlt, N. Tao, J.L. Brown, H.K. Patel response to elacestrant versus fulvestrant. Indeed, a recent report Analysis and interpretation of data (e.g., statistical analysis, biostatistics, suggested a unique ER-targeting profile for each SERM/SERD computational analysis): T. Bihani, H. Arlt, N. Tao, H. Jiang, G. Hattersley, tested (9). This might explain, in part, the differences in efficacy F. Garner, H.K. Patel observed in the models tested here in response to fulvestrant or Writing, review, and/or revision of the manuscript: T. Bihani, H. Arlt, N. Tao, elacestrant treatment. H. Jiang, J.L. Brown, D.M. Purandare, G. Hattersley, F. Garner, H.K. Patel ESR1 Administrative, technical, or material support (i.e., reporting or organizing The prevalence of mutations is gaining increasing atten- data, constructing databases): T. Bihani, H. Arlt, N. Tao tion as current advances in technology readily allow the detection Study supervision: J.L. Brown, D.M. Purandare, F. Garner, H.K. Patel of these and other mutations from circulating tumor DNA in plasma samples, highlighting their clinical importance (43). Acknowledgments Recent studies have demonstrated that patients treated with The authors thank Drs. Carlos Arteaga, Mitch Dowsett, Alison aromatase inhibitors are more likely to develop treatment-resis- O'Neill and Abhay Patki for their thorough review of the manuscript tant tumors that harbor ESR1 mutations (26, 27, 32). In fact, and helpful suggestions. The authors thank Phillips Gilmore, Oncology approximately one-third of patients previously treated with an AI Communications Inc., and Jeanne McAdara, PhD, for professional were shown to have a mutation in ESR1 (32). Some evidence in assistance with manuscript preparation, which was funded by Radius Health Inc. the literature suggests a shift in potency of fulvestrant when The costs of publication of this article were defrayed in part by the payment of evaluated in preclinical models that express ESR1 mutations page charges. This article must therefore be hereby marked advertisement in (30). However, recent data from the PALOMA-3 trial demonstrat- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ed that the presence of ESR1 mutations did not affect the extent of clinical benefit observed in fulvestrant-treated patients (32). Received October 11, 2016; revised February 10, 2017; accepted May 1, 2017; Consistent with this, we demonstrate elacestrant was able to published OnlineFirst May 4, 2017.

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OF12 Clin Cancer Res; 2017 Clinical Cancer Research

Elacestrant (RAD1901), a Selective Estrogen Receptor Degrader (SERD), Has Antitumor Activity in Multiple ER + Breast Cancer Patient-derived Xenograft Models

Teeru Bihani, Hitisha K. Patel, Heike Arlt, et al.

Clin Cancer Res Published OnlineFirst May 4, 2017.

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